Genotoxicity and morphological changes induced by the alkaloid monocrotaline, extracted from Crotalaria retusa, in a model of glial cells

Plants of Crotalaria genus (Leguminosae) present large amounts of the pyrrolizidine alkaloid monocrotaline (MCT) and cause intoxication to animals and humans. Therefore, we investigated the MCT-induced cytotoxicity, morphological changes, and oxidative and genotoxic damages to glial cells, using the human glioblastoma cell line GL-15 as a model. The comet test showed that 24 h exposure to 1-500 μM MCT and 500 μM dehydromonocrotaline (DHMC) caused significant increases in cell DNA damage index, which reached 42-64% and 53%, respectively. Cells exposed to 100-500 μM MCT also featured a contracted cytoplasm presenting thin cellular processes and vimentin destabilisation. Conversely, exposure of GL-15 cells to low concentrations of MCT (1-10 μM) clearly induced megalocytosis. Moreover, MCT also induced down regulation of MAPs, especially at the lower concentrations adopted (1-10 μM). Apoptosis was also evidenced in cells treated with 100-500 μM MCT, and a later cytotoxicity was only observed after 6 days of exposure to 500 μM MCT. The data obtained provide support for heterogenic and multipotential effects of MCT on GL-15 cells, either interfering on cell growth and cytoskeletal protein expression, or inducing DNA damage and apoptosis and suggest that the response of glial cells to this alkaloid might be related to the neurological signs observed after Crotalaria intoxication.     

Assessment of the genotoxicity of three cryoprotectants used for human oocyte vitrification: Dimethyl sulfoxide,ethylene glycol and propylene glycol

Vitrification requires high concentrations of cryoprotectants that may induce long-term toxic effects on cells. The aim of this study was to evaluate the possible genotoxicity of three cryoprotectants extensively used for oocyte vitrification: dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PROH). For this purpose, a Chinese Hamster Ovary cell line (CHO), commonly used in genetic toxicology, was selected as an in vitro biological model to assess both the induction of DNA strand-breaks as identifiable by the alkaline comet assay and the persistence of chromosomal damages (micronuclei) as analyzed by the micronucleus assay. Results showed that DMSO was not genotoxic. EG did not exert direct genotoxic activity, however EG exhibited significant genotoxic and clastogenic activities in the presence of an external cytochrome-based P450 oxidation system (S9 Mix). PrOH produced in vitro DNA-damage leading to chromosome mutations in the presence and absence of the S9 Mix. These results showed that high concentrations of EG and PrOH could induce in vitro chromosomal damage in eukaryotic cells.     

Safety evaluation of an açai-fortified fruit and berry functional juice beverage (MonaVie Active)

The safety of an açai (Euterpe oleracea Mart.) pulp enriched fruit and berry juice, MonaVie Active^®, fortified with the functional ingredient, glucosamine, was studied. The beverage was found not to be mutagenic, clastogenic, cytotoxic, or genotoxic, as determined by the bacterial reverse mutation assay, chromosomal aberration assay, mouse micronucleus assay, and mammalian cell gene mutation (L5178Y) assay. The single dose LD50 based on a 14-day acute oral toxicity study is greater than 20,000 mg/kg bw, the highest dose tested. In a repeat dose 90-day oral subchronic toxicity study by gavage, 220 animals were randomly assigned to a control group, an untreated group, or one of three experimental groups (10, 20 and 40 g/kg bw). No treatment-related significant changes in body weight, food and water consumption, ophthalmology, organ weights, urinanalysis, hematological and clinical chemistry, or gross pathology, were observed in surviving animals compared to the control groups. Three animals died midway through the observation period (male, 20 g/kg bw/day; male 40 g/kg bw/day; and, female, 10 g/kg bw/day). These animals died without preceding clinical symptoms, histopathological lesions, or evidence of injury to tissue or organs except for signs of suffocation/aspiration congestion, which was concluded to be due to problems with the gavage administration of the fluid test article, and not due to the test article itself. The NOEAL was determined to be 40 g/kg bw/day for male and female rats, which was the highest dose tested. Phylloquinone (vitamin K1) content averaged 21.7 μg/100 g, comparable to amounts found in iceberg lettuce. In conclusion, the results provide additional experimental evidence that MonaVie Active^® juice is non-toxic.     

氧化型染发剂DNA交联作用的研究

采用溴乙锭荧光法测定氧化型染发剂的主要成分对苯二胺（ＰＰＤ）、过氧化氢（Ｈ２Ｏ２）和ＰＰＤ－Ｈ２Ｏ２混合物对小牛胸腺ＤＮＡ的双链间交联形成作用。结果表明：用０～６％的对苯二胺及０～３％的Ｈ２Ｏ２对小牛胸腺ＤＮＡ直接染毒，３７℃３０ｍｉｎ时无双链间ＤＮＡ交链的形成；而ＰＰＤ和Ｈ２Ｏ２发生氧化反应后则具有双链ＤＮＡ交联形成作用并具有剂量反应关系（ｒ＝０．８２９，Ｐ＜０．０１），在受试剂量为０～０．８％（以对苯二胺含量计）时，氧化型对苯二胺的ＤＮＡ交联作用增加了４．２倍。研究结果提示，ＤＮＡ交联作用可能是氧化型染发剂遗传毒作用过程中ＤＮＡ损伤的一种方式。     

Biological activity of N-nitrosodiethanolamine and of potential metabolites which may arise after activation by alcohol dehydrogenase in Salmonella typhimurium,in mammalian cells,and in vivo

Summary The potent carcinogen N-nitrosodiethanolamine (NDELA) which is nonmutagenic in standard modifications of the S. typhimurium/mammalian microsome assay, can be activated effectively by alcohol dehydrogenase/NAD (ADH/NAD) to intermediates which are directly mutagenic in strains TA 98 and TA 100. The expected metabolites N-nitroso-2-hydroxymorpholine (NHMor), N-nitroso-(2-hydroxyethyl)-glycine (NHEG), N-nitrosoiminodiacetic acid (NIDA), and glycolaldehyde were assayed for their direct mutagenic activities in S. typhimurium TA 1535, TA 98, and TA 100. All compounds were clearly mutagenic in TA 100, but different specifities were observed for the other strains. NDELA and its putative mutagenic metabolites were also tested for induction of genotoxic activities by determination of DNA single strand breaks in primary rat hepatocytes. In these cells, NDELA and NHMor were clearly genotoxic, whereas NHEG and NIDA were inactive. In contrast, when assayed for the induction of selective DNA amplification NDELA and its metabolites were not found to induce SV40 DNA synthesis in SV40-transformed Chinese Hamster cells. The compounds were also assayed for induction of DNA single strand breaks in the liver after a single oral application to rats. NDELA and NHMor were about equally active in this in vivo test, whereas NHEG, NIDA and glycolaldehyde were inactive. Differences in biological activity in the cultivated cells, as compared to hepatocytes or to the in vivo situation may most probably be due to differences in metabolism and/or pharmacokinetics.Dedicated to Professor Dietrich Schmähl on the occasion of his 60th birthday     

Micronucleus induction in cells co-exposed in vitro to 50 Hz magnetic field and benzene, 1,4-benzenediol (hydroquinone) or 1,2,4-benzenetriol

The generation, transmission (e.g. power lines, transformers, service wires, and electrical panels), and use (e.g. home appliances, such as electric blankets, shavers, and televisions) of electrical energy is associated with the production of weak electric and magnetic fields (EMF) which oscillate 50 (Europe) or 60 (USA) times per second (power-line frequency), falling in the extremely-low frequency (ELF) region of the electromagnetic spectrum. Epidemiological reports suggest a possible association between exposure to ELF-EMF and an increased risk of cancer (e.g. childhood acute leukaemia). Benzene is an established human leukomogen. This xenobiotic, which is unlikely to be the ultimate carcinogen, is metabolized in the liver to its primary metabolite phenol, which is hydroxylated to hydroquinone (1,4-benzenediol) and 1,2,4-benzenetriol. In this in vitro approach, to test the genotoxic and / or co-genotoxic potency of ELF-EMF, the cytokinesis block micronucleus (MN) method with Jurkat cells has been used. A 50 Hz magnetic field (MF) of 5 mT field strength was applied for different length of time (from 1 to 24 h), either alone or with benzene, 1,4-benzenediol, or 1,2,4-benzenetriol. Our preliminary results show that, after 24 h exposure, the frequency of micronucleated cells in MF-exposed cultures is 1.9 fold higher than in sham-exposed (control) cultures. Benzene exposure does not show any cytogenetic activity, whereas 1,4-benzenediol or 1,2,4-benzenetriol alone significantly affect the number of MN in Jurkat cells, as compared to untreated cultures. Moreover, co-exposure to ELF-MF does not seem to affect the frequency of micronuclei induced by benzene, 1,4-benzenediol, or 1,2,4-benzenetriol.     

N-Nitrosodiethanolamine is activated in the rat to an ultimate genotoxic metabolite by sulfotransferase

Summary Inhibition of sulfotransferase by 2,6-dichloro-4-nitrophenol (DCNP) has been found to completely abolish the genotoxic potential of N-nitrosodiethanolamine (NDELA) in rat liver as indicated by induction of DNA single strand breaks. The DNA strand breaking potential of N-nitroso-2-hydroxymorpholine (NHMOR), a metabolite of NDELA formed by alcohol dehydrogenase-mediated oxidation, was also almost quantitatively abolished. In contrast to these -hydroxylated nitrosamines, the effectiveness of N-nitrosodiethylamine (NDEA) remained unaffected by DCNP with respect to its DNA damaging potential. N-Nitrosoethylethanolamine (NEELA) was the most potent genotoxic agent of this series of nitrosamines and its strand breaking activity was only partialy inhibited by DCNP. A new activation mechanism for NDELA is proposed: NDELA is transformed at first by alcohol dehydrogenase into the cyclic hemiacetal NHMOR. This cyclic -hydroxynitrosamine appears to be a substrate for sulfotransferase. The resulting sulfate conjugate is suggested to be the ultimate genotoxic electrophile. However, the results do not exclude the possiblity that NDELA itself undergoes sulfate conjugation.Dedicated to Professor Dietrich Schmähl on the occasion of his 60th birthday     

Passive smoking at work: biochemical and biological measures of exposure to environmental tobacco smoke

Summary Several biochemical and biological measures of tobacco smoke intake were used to evaluate exposure of restaurant personnel to environmental tobacco smoke as compared with active smokers and non-exposed non-smokers. All of the measured parameters — carboxyhaemoglobin (COHb), thiocyanate (SCN) and cotinine in plasma, cotinine and mutagenicity in urine, total white blood cell count (WBC), and sister chromatid exchange (SCE) frequency in cultured lymphocytes — were significantly elevated in the smoker group (n = 22) compared to the non-exposed group (n = 20). Work-related passive exposure (n = 27) was seen most clearly in the cotinine values, both from plasma (mean P-cot in passive smokers 10 ng/ml vs 5.2 ng/ml in non-exposed) and from urine (mean U-cot in passive smokers 56 ng/ml vs 8.3 ng/ml in non-exposed), but significant increases were also seen in the thiocyanate levels (mean P-SNC in passive smokers 58 mol/1 vs 46 mol/1 in non-exposed) and, as a preliminary finding, in total leucocyte count (in passive smokers 8.0 × 10⁹/1 vs 6.8 x 10⁹/1 in non-exposed). The results demonstrate that environmental tobacco smoke may be an occupational health hazard.     

Black bean (Phaseolus vulgaris L.) as a protective agent against DNA damage in mice.

This study was designed to evaluate the toxicogenetic or protective effect of cooked and dehydrated black beans (Phaseolus vulgaris L.) in bone marrow and peripheral blood cells of exposed mice. The frequency of micronuclei detected using the bone marrow erythrocyte micronucleus test and level of DNA lesions detected by the comet assay were chosen as end-points reflecting mutagenic and genotoxic damage, respectively. Initially, Swiss male mice were fed with a 20% black bean diet in order to detect mutagenic and genotoxic activity. However, no increase in the frequency of bone marrow micronucleated polychromatic erythrocytes (MN PCEs) or DNA lesion in leukocytes was observed. In contrast, received diets containing 1, 10 or 20% of black beans, a clear, but not dose-dependent reduction in the frequency of MN PCEs were observed in animals simultaneously treated with cyclophosphamide, an indirect acting mutagen. Similar results were observed in leukocytes by the comet assay. Commercial anthocyanin was also tested in an attempt to identify the bean components responsible for this protective effect. However, instead of being protective, the flavonoid, at the highest dose administered (50 mg/kg bw), induced primary DNA lesion, as detected by the comet assay. These data indicate the importance of food components in preventing genetic damage induced by chemical mutagens, and also reinforce the role of toxicogenetic techniques in protecting human health.     

Black bean (Phaseolus vulgaris L.) as a protective agent against DNA damage in mice

This study was designed to evaluate the toxicogenetic or protective effect of cooked and dehydrated black beans (Phaseolus vulgaris L.) in bone marrow and peripheral blood cells of exposed mice. The frequency of micronuclei detected using the bone marrow erythrocyte micronucleus test and level of DNA lesions detected by the comet assay were chosen as end-points reflecting mutagenic and genotoxic damage, respectively. Initially, Swiss male mice were fed with a 20% black bean diet in order to detect mutagenic and genotoxic activity. However, no increase in the frequency of bone marrow micronucleated polychromatic erythrocytes (MN PCEs) or DNA lesion in leukocytes was observed. In contrast, received diets containing 1, 10 or 20% of black beans, a clear, but not dose-dependent reduction in the frequency of MN PCEs were observed in animals simultaneously treated with cyclophosphamide, an indirect acting mutagen. Similar results were observed in leukocytes by the comet assay. Commercial anthocyanin was also tested in an attempt to identify the bean components responsible for this protective effect. However, instead of being protective, the flavonoid, at the highest dose administered (50 mg/kg bw), induced primary DNA lesion, as detected by the comet assay. These data indicate the importance of food components in preventing genetic damage induced by chemical mutagens, and also reinforce the role of toxicogenetic techniques in protecting human health.